Shreffler:Notebook/Alex Daily/2011/05/19

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Buhlmann Assay pilot

Ran Buhlmann assay for first time using buffy coats from Tom Kadlik (RBC) and MGH. Used five dilutions each of Milk and Peanut allergen (1.00, 0.20, 0.040, 0.0080, 0.0016 µg/mL)

Followed Buhlmann protocol with following differences:

  • Used buffy coat rather than whole blood, diluted with RPMI to 60% of original constitution
  • Incubation step in incubator rather than water bath
  • After lysis, resuspended pellet in lab-made staining buffer (0.5% BSA, 2 mM EDTA) rather than kit-provided wash buffer
  • Blood used was one day old (both buffy coats were drawn 5/18/11)
  • Flow acquisition was done following day (5/19/11), after being left overnight in staining buffer


Observations/notes

Heavy clumping in both the Kadlik and MGH samples, possibly due to sitting in staining buffer overnight. Kadlik samples were jettisoned due to low basophil event count. MGH 5/18/11 basophils were nonresponding to anti-FCeRI Ab - next time, should also include anti-IgE Ab as additional positive control. Samples were unsensitized to both milk and peanut series.